We believe this site might serve you best:

United States

Select a Different Country and Language

Promega's Cookie Policy

Our website uses functional cookies that do not collect any personal information or track your browsing activity. When you select your country, you agree that we can place these functional cookies on your device.

SEARCH ARTICLES

Related Protocols

Related Resources

Abstract

Promega HisLink™ Protein Purification Systems (Cat.# V8821, V3680 and V3681) provide a reliable and easy method for the purification of polyhistidine and HQ-tagged proteins directly from a crude lysate in either a manual or an automated format. Depending on the system used it is possible to purify tagged proteins using large-scale or high-throughput methods. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry is a valuable tool for analyzing purified proteins. However, purified polyhistidine-tagged protein samples often contain materials that are not compatible for analysis with MALDI-TOF mass spectrometry. Previously we have described a method of eluting polyhistidine-tagged proteins using the MagneHis™ Protein Purification System (Cat.# V8550) for analysis using mass spectrometry. In this article, we show that the polyhistidine-tagged proteins can be eluted from HisLink™ Protein Purification Resin using 40% ethanol or 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile. These samples can be directly analyzed by MALDI-TOF mass spectrometry and produce extremely clean data. The elution conditions reported in this article can be used for the high-throughput MALDI-TOF MS analysis of polyhistidine-tagged proteins.

Laurie Engel, Becky Godat, Rod Flemming and Tonny Johnson

Promega CorporationPublication Date: February 2006

Introduction

Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and other alternative methods of mass spectrometry (MS) analysis have become essential methods of protein analysis(1)(2)
. Using MS methods researchers identify proteins, peptides(3)(4)
, post-translational modifications(5)(6)(7)
, protein profiles, protein:protein interactions, protein:small molecule interactions(8)(9)
and study protein structure and function(10)
. Polyhistidine-tagged protein purification systems provide large amounts of a protein or small amounts of multiple proteins for study. However, the elution buffers used in these systems contain salts (e.g., imidazole) that cannot be used in mass spectrometry analysis. To be compatible with MALDI–TOF MS analysis, eluted samples need to undergo tedious dialysis methods or size exclusion separation techniques to remove salts. Previously we have described a method of eluting polyhistidine-tagged proteins using the MagneHis™ Protein Purification System (Cat.# V8550) for analysis using mass spectrometry(11)
. In this article, we describe a method for the elution of a polyhistidine-tagged protein from HisLink™ Protein Purification Resin using 40% ethanol or 0.1% TFA in 50% acetonitrile. These elution conditions allow direct MS analysis and provide clean mass spectrometry data necessary for high-throughput analysis using MALDI-TOF MS.

Polyhistidine-tagged calmodulin was purified from E. coli strain BL-21 (DE3) using HisLink™ Resin as described in the HisLink™ 96 Protein Purification System Technical Bulletin (#TB342). Wash and elution conditions were optimized to decrease substances that interfere with mass spectrometry analysis. The resin/protein complex was first washed twice with 500µl of 100mM HEPES (pH 7.5) plus 0.5M NaCl to decrease nonspecific binding to the resin. The particles were then washed four times with Nuclease Free Water (Cat.# P1193) to remove the NaCl and buffer from the resin. The protein was eluted from the resin using either 0.1% TFA in 50% acetonitrile or 40% ethanol. Samples were dried using a SpeedVac® DNA 110 Concentrator and were resuspended in 200µl of Nuclease-Free Water. A portion of the rehydrated samples was removed for protein gel analysis, and the remainder was dried and sent to HT Laboratories (San Diego, CA) for MALDI-TOF MS analysis.

Results and Conclusions

Gel analysis showed that the protein of interest (His-calmoulin) was eluted from the resin using the various elution conditions (Figure 1). The amounts of protein recovered using the alternative methods appear to be less than the MagneHis™ Elution Buffer (control); however, the protein amount is sufficient for MALDI-TOF MS analysis. MALDI-TOF MS data show the MagneHis™ Elution Buffer (containing imidazole) causes extensive background interference (Figure 2); however the samples eluted with 40% ethanol or 0.1% TFA in 50% acetonitrile show clean protein peaks well above background (Figures 3 and 4).

Scientists at Your Service

We offer a range of services to help you succeed using Promega technologies. From product training to set up of automated systems and development of custom applications—our scientific support goes beyond the basics.